Direct immunoassay for detecting Escherichia coli colonies that contain polypeptides encoded by cloned DNA segments.
We describe a simple in situ immunoassay for screening Escherichia coli colonies, to detect those that express polypeptide antigens encoded by cloned DNA segments. In the colony immunoassay that we have developed, only one antibody molecule need bind to the polypeptide molecule. Hence short fragments of coding sequences should be detectable, and problems imposed by the conformation of polypeptides "fused" to protein fragments encoded by the vector should be minimized. The method should allow the use of monoclonal antibodies, which bind to only one determinant. Colonies grown on nitrocellulose filters were lysed in situ and proteins from the lysates were blotted onto CNBr-activated filter papers. Antigens covalently bound to the CNBr paper were detected by reaction with antiserum, followed by 125I-labeled protein A from Staphylococcus aureus and autoradiography. We have used the colony immunoassay to determine what proportion of ptrpED5-1 clones containing mouse mu chain cDNA segments express mu polypeptides. Surprisingly, about 50% of the clones were positive. Because only one in six clones would be expected to contain the insert in the correct orientation and reading frame to encode a fused polypeptide, it is likely that polypeptides that initiate within the insert can be detected. A similarly high proportion of pBR322 clones containing mu cDNA were positive, and about 25% of pBR322 clones containing mouse gamma 2a chain cDNA were positive to anti-gamma 2a serum. EAch of the positive mu clones reacted with affinity-purified anti-mu antibodies but not with normal serum, and clones containing irrelevant sequences were negative. The mu cDNA segment was inserted in the correct orientation in all six clones studied, and discrete mu polypeptides corresponding in size both to fused polypeptides and internally initiated polypeptides were detected by electrophoresis of protein extracts.
D003001:Cloning, Molecular / Q000379:methods*; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics* / Q000276:immunology; D005796:Genes; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007148:Immunoglobulin mu-Chains / Q000235:genetics*; D007158:Immunologic Techniques; D009876:Operon; D010957:Plasmids; D014364:Tryptophan / Q000235:genetics
D004274:DNA, Recombinant; D007143:Immunoglobulin Heavy Chains; D007148:Immunoglobulin mu-Chains; D014364:Tryptophan